In the field of molecular diagnostics, the detection of target nucleic acids with the polymerase chain reaction (PCR) plays an important role. The routine screening of blood banks for the presence of Human Immunodeficiency Virus (HIV), Hepatitis-B (HBV) or C Virus (HCV) is an example for the large-scale application of PCR-based diagnostics. Automated systems for PCR-based analysis often make use of real-time detection of product amplification during the PCR process. Key to such methods is the use of modified oligonucleotides carrying reporter groups or labels.
In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific nucleic acid sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the target sequence that is the region of interest in the target nucleic acid. A repetitive series of reaction steps involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase (DNA: deoxyribonucleic acid) results in the exponential accumulation of a specific fragment whose termini are defined by the 5′ ends of the primers.
The detection of DNA amplification products generated by a PCR process can, on the one hand, be accomplished in separate working steps. These may involve the characterization of amplified fragments with respect to their electrophoretic mobility and/or the analysis of denatured amplification products attached to a solid support using a hybridization probe.
On the other hand, the detection of DNA amplification products can be done in a so-called “homogeneous” assay system. A “homogeneous” assay system comprises reporter molecules or labels which generate a signal while the target sequence is amplified. An example for a “homogeneous” assay system is the TaqMan® system that has been detailed in U.S. Pat. Nos. 5,210,015, 5,804,375 and 5,487,972. Briefly, the method is based on a double-labeled probe and the 5′-3′ exonuclease activity of Taq DNA polymerase. The probe is complementary to the target sequence to be amplified by the PCR process and is located between the two PCR primers during each polymerization cycle step. The probe has two fluorescent labels attached to it. One is a reporter dye, such as 6-carboxyfluorescein (FAM), which has its emission spectra quenched by energy transfer due to the spatial proximity of a second fluorescent dye, 6-carboxy-tetramethyl-rhodamine (TAMRA). In the course of each amplification cycle, the Taq DNA polymerase in the process of elongating a primed DNA strand displaces and degrades the annealed probe, the latter due to the intrinsic 5′-3′ exonuclease activity of the polymerase. The mechanism also frees the reporter dye from the quenching activity of TAMRA. As a consequence, the fluorescent activity increases with an increase in cleavage of the probe, which is proportional to the amount of PCR product formed. Accordingly, amplified target sequence is measured detecting the intensity of released fluorescence label.
A similar principle of energy transfer between fluorescent dye molecules applies to “homogeneous” assays using so-called “molecular beacons” (U.S. Pat. No. 6,103,476). These are hairpin-shaped nucleic acid molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid (U.S. Pat. No. 6,103,476). They are designed in such a way that the loop portion of the molecule is a probe sequence complementary to a region within the target sequence of the PCR process. The stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence. A fluorescent moiety is attached to the end of one arm and a quenching moiety is attached to the end of the other arm. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. Since the quencher moiety is a non-fluorescent chromophore and emits the energy that it receives from the fluorophore as heat, the probe is unable to fluoresce. When the probe encounters a target molecule, it forms a hybrid that is longer and more stable than the stem hybrid and its rigidity and length preclude the simultaneous existence of the stem hybrid. Thus, the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem apart, and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence which can be detected.
More examples for “homogeneous” assay systems are provided by the formats used in the LightCycler® instrument (see e.g. U.S. Pat. No. 6,174,670), some of them sometimes called “kissing probe” formats. Again, the principle is based on two interacting dyes which, however, are characterized in that the donor-dye excites an acceptor-dye by fluorescence resonance energy transfer. An exemplified method uses two modified oligonucleotides as hybridization probes, which hybridize to adjacent internal sequences of the target sequence of the PCR process. The 5′-located modified oligonucleotide has a donor-dye as a label at its 3′ end. The 3′-located modified oligonucleotide has an acceptor-dye at its 5′ end. Following the head-to-tail-oriented annealing of the two modified oligonucleotides to the target sequence in the course of an amplification cycle, donor and acceptor dye are brought in close proximity. Upon specific excitation of the donor dye by means of a monochromatic light pulse, acceptor dye fluorescence is detected providing a measure for the amount of PCR product formed.
Another assay format is the so-called “array” format. An “array” is an arrangement of addressable locations on a device (see e.g. U.S. Pat. Nos. 5,143,854, 6,022,963, 6,156,501, WO 90/15070, WO 92/10092). The number of locations can range from several to at least hundreds of thousands. Most importantly, each location represents a totally independent reaction site. Each location carries a nucleic acid as e.g. an “oligomeric compound”, which can serve as a binding partner for a second nucleic acid, in particular a target nucleic acid. Methods for the manufacturing thereof are described in EP-A-0 476 014 and Hoheisel, J. D., TIBTECH (1997), Vol. 15,465-469, WO 89/10977, WO 89/11548, U.S. Pat. Nos. 5,202,231, 5,002,867, WO 93/17126). Further development has provided methods for making very large arrays of oligonucleotide probes in very small areas.(U.S. Pat. No. 5,143,854, WO 90/15070, WO 92/10092). Microfabricated arrays of large numbers of oligonucleotide probes, called “DNA chips” offer great promise for a wide variety of applications (see e.g. U.S. Pat. Nos. 6,156,501 and 6,022,963). The basic steps of the method are that nucleic acid from control and treatment samples is isolated and labeled with different fluorescent dyes incorporated during an amplification process. In more detail, this is performed according to the method described in U.S. Pat. Nos. 5,545,522; 5,716,785; 5,891,636; 6,291,170 whereby double stranded cDNA is synthesized with a primer comprising the bacterial T7-Promoter and labeled RNA is transcribed in the presence of ribonucleoside triphosphates whereby labels are attached to some of the nucleoside triphosphates. These labeled nucleic acids are then optionally fragmented, mixed and hybridized to the arrayed oligomeric compounds. An optical device is then used to measure the relative intensities of each dye for each individual spot. The ratio of fluorescence levels between the two probes indicates the relative gene expression between the samples. By these processes researchers can evaluate an entire set of genes simultaneously rather than looking at the effects of single genes one at a time. High differential expression of specific genes can then be followed up by conventional means such as northern blot or quantitative RT-PCR. Data from multiple experiments can be combined in order to assign functional information to genes of otherwise unknown function. Genes showing similar expression profiles across differing states are likely to participate in common physiological or metabolic pathways. Cluster analysis programs have been developed which allow detection of co-expressed groups of genes reflecting information on function.
The oligomeric compound or modified oligonucleotide used in “homogeneous” assay systems comprises nucleotides, modified nucleotides or non-nucleotide compounds, i.e. the monomeric units, to which labels such as dyes as reporter molecules are attached. The features of such monomeric units are that they can be attached to and/or integrated into the sugar-phosphate polymer backbone of a nucleic acid, they do not prevent the pairing of the modified oligonucleotide with its complementary target sequence, and they provide functional groups for the attachment of one or more labels.
These requirements can be fulfilled by nucleotides, modified nucleotides or non-nucleotide compounds. In addition, the TaqMan® format requires that the oligomeric compound can be digested by 5′-3′ exonuclease activity of a template-dependent DNA-polymerase. Various modified nucleotides have also been incorporated into oligomeric compounds to influence their hybridization behavior or stability, see e.g. WO02/12263.
Several compounds and their use for incorporation as monomeric units into nucleic acids are known in the art. Such compounds provide functional groups and/or linking moieties for the covalent attachment of reporter groups or labels. In the course of the chemical synthesis of the oligomeric compound, the skeletal structure of the “non-nucleotide compound” or “modified nucleotide” is connected with the “oligonucleotide” backbone, for example by phosphoramidite-based chemistry resulting in a phosphodiester. A given incorporated compound thus represents a “modified nucleotide” or “non-nucleotide compound” within the newly generated “modified oligonucleotide”. A label is bound by a reactive group of a linking moiety, exemplified by but not limited to an amino function that is present on the skeletal structure or on the “linking moiety”, which connects the skeleton with the reactive group. A label can be covalently attached to the compound prior to the synthesis of a “modified oligonucleotide” or afterwards, upon the removal of an optional protecting group from the functional group to which the label is to be coupled. Various “modified nucleotides” have also been incorporated into oligomeric compounds to influence their hybridization behavior or stability, see e.g. WO02/12263.
Several references disclose modified nucleotides comprising six-membered rings and their incorporation into oligonucleotides. WO 93/25565 discloses 1,5 anhydrohexitol nucleoside analogs and pharmaceutical uses thereof. Further 1,5 anhydrohexitol or hexitol nucleoside analogues are disclosed in Verheggen, I., et al., Nucleosides & Nucleotides 15 (1996) 325-335; Verheggen, I., et al., J. of Med. Chem. 36 (1993) 2033-2040; Pérez-Pérez, M.-J., et al., Bioorg. & Med. Chem. Letters 6 (1996) 1457-1460; Vastmans, K., et al., Collect. Symp. Series 2 (1999) 156-160; Andersen, M. W., et al., Pergamon, Tetrahedron Lett. 37 (1996) 8147-8150; Ostrowski, T., et al., J. Med. Chem. 41 (1998) 4343-4353; Allart, B., et al., Tetrahedron 55 (1999) 6527-6546; De Bouvere, B., et al., Liebigs Ann./Recueil (1997) 1453-1461; Verheggen, I., J. Med. Chem. 38 (1995) 826-835. Antiviral tetrahydropyrans are disclosed in U.S. Pat. No. 5,314,893. A deoxyadenosine bisphosphate derivative containing a six-membered ring is described by Brown, S. G., et al., Drug Development Research 49 (2000) 253-259.
WO 01/18003 describes six-membered at least partially unsaturated carbocyclic nucleoside compounds. Pharmaceutical uses are primarily considered. Further cyclohexane or cyclohexene containing nucleoside analogs are disclosed in Wang, J., et al., J. Med. Chem. 43 (2000) 736-745; Rosenquist, A., et al., J. Org. Chem. 61 (1996) 6282-6288; Ramesh, K., et al., J. Org. Chem. 57 (1992) 5861-5868; Pérez-Pérez, M. J., et al., J. Org. Chem. 60 (1995) 1531-1537; Konkel, M. J., and Vince, R., Tetrahedron 52 (1996) 799-808; Arango, J. H., Nucleosides & Nucleotides 12 (1993) 773-784; Konkel, M. J., and Vince, R., Nucleosides & Nucleotides 14 (1995) 2061-2077; Katagiri, N., et al., Nucleosides & Nucleotides 15 (1996) 631-647; Luyten, I., and Herdewijn, P., Tetrahedron 52 (1996) 9249-9262; Wang, J., et al., J. Med. Chem. 43 (2000) 736-745; Wang, J., and Herdewijn, P., J. Org. Chem. 64 (1999) 7820-7827; Maurinsh, Y., et al., J. Org. Chem. 62 (1997) 2861-2871; Wang, J., et al. Nucleosides Nucleotides Nucleic Acids 20 (2001) 785-788; Maurinsh, Y, et al., Chem. Eur. J. 5 (1999) 2139-2150.
WO 96/05213 discloses oligomers consisting or comprising 1,5 anhydrohexitol nucleoside analogues and their uses. WO97/30064 relates to oligomers comprising or containing 1,5-anhydrohexitol nucleotide analogs which exhibit sequence specific binding to complementary sequences. Further, the synthesis thereof is disclosed and their use in diagnosis, hybridization, isolation of nucleic acids, site-specific DNA modification and therapeutics. Further hexitol or 1,5 anhydrohexitol containing nucleic acids are disclosed in Vastmans, K., et al., Biochem. 39 (2000) 12757-12765; Pochet, S., et al., Nucleosides & Nucleotides 18 (1999) 1015-1017; Kozlov, I. A., et al., Chemistry 6 (2000) 151-155; Vandermeeren, M., et al., Biochem. Pharm. 59 (2000) 655-663; Hendrix, C., et al., Chem. Eur. J. 3 (1997) 1513-1520; Allart, B., et al., Chem. Eur. J. 8 (1999) 2424-2431; Kozlov, I. A., et al., J. Am. Chem. Soc. 121 (1999) 2653-2656; Hendrix, C., et al., Chem. Eur. J. 3 (1997) 110-120; Van Aerschot, A., et al., Angew. Chem. Int. Ed. Engl. 34 (1995) 1338-1339; Froeyen, M., et al., Helvetica Chimica Acta 83 (2000) 2153-2182; Boudou, V., et al., Nucleic Acids Research 27 (1999) 1450-1456; Lescrinier, E., et al., Chem. Biol. 7 (2000) 719-731; Kozlov, I. A., et al., J. Am. Chem. Soc. 121 (1999) 1108-1109; Herdewijn, P., et al., Nucl. Acids Symp. Series 31 (1994) 161-162; Lescrinier, E., et al., Helvetica Chimica Acta 83 (2000) 1291-1310; Kozlov, I. A., et al., J. Am. Chem. Soc. 121 (1999) 5856-5859; De Winter, H., et al., J. Am. Chem. Soc. 120 (1998) 5381-5394; Atkins, D., et al., Pharmazie 55 (2000) 615-617. Pyranosyl oligomers are also disclosed in WO 98/25943 and WO 99/15509.
WO 01/49687 discloses cyclohexene nucleic acids, their hybridization behavior to RNA and the use in diagnostics and therapy. Further cyclohexane or cyclohexene containing nucleic acids are disclosed in Wang, J., et al., J. Am. Chem. Soc. 122 (2000) 8595-8602; Maurinsh, Y., et al., Chem. Eur. J. 5 (1999) 2139-2150.
WO97/27317 and WO 02/072779 disclose various compounds for nucleic acid labeling.
EP 0468352 discloses nucleic acid derivatives comprising a general formula that contains a six-membered ring but which contain an additional methylene group at the C4 atom of the six-membered ring. WO01/02417 and Jung, K.-E., Bioorg. Med. Chem. Lett. 9 (1999) 3407-3410 disclose a nucleotide monomer containing a six-membered aza-sugar and oligomers containing them.
Compounds to be used for the incorporation of labels into nucleic acids or to influence the properties of the modified oligonucleotide have to be carefully selected as they may interfere with base pairing, fail to provide a skeletal structure of sufficient rigidity, provide largely hydrophobic structures resulting in low water solubility, provide only limited amenability to chemical modifications, or comprise mixtures of enantiomers. Therefore, it was an object of the present invention to provide new compounds to be used for the incorporation of labels into nucleic acids or to influence their properties.